Summary of Work: The physiological correlates of the major stages in the morphogenesis of the mammalian central nervous system, including proliferation, apoptosis, migration and differentiation remain to be elucidated. Relatively few extramural research programs are aimed in this direction, although the field is growing. We have used electrophysiological and digital videomicroscopic recording techniques, which complement those employed in Z01 NS 02230-22 (Biological properties developing in central nervous system cells), to study the development, differentiation and cellular distribution of physiological properties expressed by progenitor and differentiating embryonic rat central nervous system cells in culture. One major project line involves the role of astrocytes in neuronal differentiation. We have discovered that the differentiating effects of astrocyte secretions involve neuronal GABA and glutamate receptors. Electrical and optical recordings of neurons revealed that astrocytes also accelerated the emergence of membrane conductances and increases in the chloride ion gradient. Differentiating neurons exhibited a chloride ion baseline signal, which could be "puffed away" using saline applied to the cell surface. The tonic signal was due to GABA?s random activation of GABA(A) receptor/chloride channels. Astrocytes enhanced the signal five-fold by both indirect and direct effects. Astrocytes also accelerated the appearance of spontaneous and evoked GABAergic, and later, glutamatergic synaptic-like transients. Astrocytes are known to synthesize and secrete a diverse array of soluble substances, some or all of which might interact with neurons. For example, astrocytes synthesize and secrete steroid metabolites. We found that the steroid metabolite previously shown to be active at GABA(A) receptor/chloride channels on differentiated neurons induced the appearance of tonic GABAergic signaling. This novel action was dose-dependent, graded in effect and stereospecific. Neither calcium-free saline nor BAPTA-AM affected the phenomenology. However, sodium-free saline and pharmacological antagonism of GABA uptake markedly depressed this effect, implicating GABA transporter activity. Potassium (7.5-20mM) also induced tonic GABAergic signaling. Fluctuation analyses of the tonic GABAergic signals demonstrated short- and long-lasting openings, whose synchronized activation accounted for the short and long transient decays. These openings may reflect different GABA(A) receptor/chloride channel subunit constructions. The mechanism(s) underlying transient GABA release remain to be elucidated. During synaptogenesis, extracellular targets of the neurotoxin tetanus toxin become localized intracellularly at pre-synaptic terminals where they line the lumen of transmitter-containing vesicles. FACS studies and confocal microscopy of tetanus toxin binding to cultured neurons confirmed the surface expression of targets (for details, see Z01 NS 02330-21). FACS analysis also revealed tetanus toxin labeling and surface GABA co-expression over ~100-fold ranges of signal intensities in neurons and in liposomes doped with a known toxin target, the ganglioside GT1b, and GABA, while confocal study resolved their co-expression in surface patches. Electrical recordings revealed that ruthenium red, which binds to sialic acids, immediately and irreversibly disturbed tonic and transient GABAergic signaling. GABA may interact with GT1b via binding to sialic acids. Synchronized detachment of GABA from these surface-accessible sites could generate the synaptic transient. BAPTA-AM, which extinguishes cytoplasmic calcium transients, quickly eliminated GABAergic transients, while tonic signaling remained. Hence, synchronized detachment of GABA from the cell surface requires cytoplasmic calcium transients, which could provide the necessary charge movement at the membrane to detach GABA in an electrostatic manner.